Potent preclinical impact of metronomic low-dose oral topotecan combined with the antiangiogenic drug pazopanib for the treatment of ovarian cancer

Abstract

Low-dose metronomic chemotherapy has shown promising activity in many preclinical and some phase II clinical studies involving various tumor types. To evaluate further the potential therapeutic impact of metronomic chemotherapy for ovarian cancer, we developed a preclinical model of advanced human ovarian cancer and tested various low-dose metronomic chemotherapy regimens alone or in concurrent combination with an antiangiogenic drug, pazopanib. Clones of the SKOV-3 human ovarian carcinoma cell line expressing a secretable beta-subunit of human choriogonadotropic (beta-hCG) protein and firefly luciferase were generated and evaluated for growth after orthotopic (i.p.) injection into severe combined immunodeficient mice; a highly aggressive clone, SKOV-3-13, was selected for further study. Mice were treated beginning 10 to 14 days after injection of cells when evidence of carcinomatosis-like disease in the peritoneum was established as assessed by imaging analysis. Chemotherapy drugs tested for initial experiments included oral cyclophosphamide, injected irinotecan or paclitaxel alone or in doublet combinations with cyclophosphamide; the results indicated that metronomic cyclophosphamide had no antitumor activity whereas metronomic irinotecan had potent activity. We therefore tested an oral topoisomerase-1 inhibitor, oral topotecan, at optimal biological dose of 1 mg/kg/d. Metronomic oral topotecan showed excellent antitumor activity, the extent of which was significantly enhanced by concurrent pazopanib, which itself had only modest activity, with 100% survival values of the drug combination after six months of continuous therapy. In conclusion, oral topotecan may be an ideal agent to consider for clinical trial assessment of metronomic chemotherapy for ovarian cancer, especially when combined with an antiangiogenic drug targeting the vascular endothelial growth factor pathway, such as pazopanib. Mol Cancer Ther; 9(4); 996-1006. (c)2010 AACR.

Figure 1. Development of an advanced orthotopic model of ovarian cancer in mice

(A) Clones of the SKOV-3 cell with stable expression of luciferase and β-hCG were injected intraperitoneally into CB-17 SCID mice as advanced orthotopic ovarian cancer model. Tumor growth was monitored by total bioluminescence image after luciferin administration. The most aggressive of these clones, SKOV-3-13, develops more peritoneal carcinomatosis and was subsequently chosen for use in all of the therapy experiments. (B) Correlation between the tumor measurement methods. i) urine β-hCG levels. Urine β-hCG levels were measured by ELISA and corrected by urine creatinine levels. When the subcutaneous tumor reached approximately 100mm³ urinary β-hCG levels (93±50 mIU/mg) were almost same as the orthotopic model the time of treatment initiation (104±43 mIU/mg). ii) Relationship between urinary β-hCG levels and bioluminescence (photons/sec) in the orthotopic transplant model, or iii) urinary β-hCG levels and tumor volume (mm³) in the subcutaneous transplant model. The solid line represents a simple linear regression between the two methods. Each dot represents an individual mouse.

Figure 2. Preliminary assessment of combination metronomic chemotherapy regimens using cyclophosphamide (CTX)

(A) Tumor growth monitored by total bioluminescence: SKOV-3-13 human ovarian cancer cells were injected i.p. into CB17 SCID mice; 10 days after tumor injection, the various treatments were initiated, including vehicle control, 100 mg/kg bolus CTX i.p. followed by 20 mg/kg cyclophosphamide daily administered through the drinking water, and combinations of metronomic CTX+ PTX 1mg/kg three times a week i.p., CTX+CPT-11 10 mg/kg twice a week i.p. and CTX+CDDP 1 mg/kg twice a week i.p. Mice were imaging every week and tumor growth was monitored by total body bioluminescence. (B) Survival curve: mice were euthanized when body weight loss exceeded 15% or mice became moribund, and assessed; n=4, bars; +SD

Figure 3. Effect of metronomic cyclophosphamide alone or in combination with irinotecan

SKOV-3-13 human ovarian cancer cells were injected i.p. into CB17 SCID mice. 14 days after tumor injection, treatment was initiated and tumor growth was monitored by total body bioluminescence. The groups were: vehicle control, 100 mg/kg initial CTX treatment, and every 6 weeks bolus CTX i.p. followed by 20 mg/kg/d CTX through the drinking water, CPT-11 10 mg/kg twice a week i.p. and combination CTX+CPT-11. (A) Tumor growth. (B) Effect on survival. Mice were euthanized when more than 20% body weight loss occurred or when moribund and then assessed. n=8, bars; +SD

Figure 4. CEP analysis to determine the optimal biologic dose of oral topotecan and pazopanib

Normal Balb/c mice were treated with oral topotecan or pazopanib. Doses used were 0, 0.25, 0.5, 1.0, 2.0 mg/kg/d daily gavage for oral topotecan, 0,10, 25,50,100 mg/kg twice a day, by gavage, and 0, 50, 100, 120, 150 mg/kg once a day, by gavage for pazopanib. CEP analysis was performed after 7 or 28 days of treatment.

Figure 5. Effect of metronomic oral topotecan alone or in combination with pazopanib

SKOV-3-13 human ovarian cancer cells were injected i.p. into CB-17 SCID mice. Treatments were initiated 14 days after tumor injection. The drug treatments and doses tested were: vehicle control, MTD topotecan (1.5mg/kg i.p. 5 consecutive days every 3 weeks), low-dose metronomic oral topotecan (1mg/kg by gavage, daily), once a day pazopanib (150mg/kg by gavage daily), twice a day pazopanib (25mg/kg by gavage), pazopanib once a day + oral topotecan and pazopanib twice a day + oral topotecan. Mice were euthanized when more than 20% body weight loss occurred or when moribund. (A) Mice were imaged every week and tumor growth bioluminescence. i) tumor growth curve,. ii) total body luminescence change over time. (B) Effect on survival. (C) Evaluate the toxicity. i) body weight, ii) WBC count. n=5, bars; +SD

Figure 5. Effect of metronomic oral topotecan alone or in combination with pazopanib

SKOV-3-13 human ovarian cancer cells were injected i.p. into CB-17 SCID mice. Treatments were initiated 14 days after tumor injection. The drug treatments and doses tested were: vehicle control, MTD topotecan (1.5mg/kg i.p. 5 consecutive days every 3 weeks), low-dose metronomic oral topotecan (1mg/kg by gavage, daily), once a day pazopanib (150mg/kg by gavage daily), twice a day pazopanib (25mg/kg by gavage), pazopanib once a day + oral topotecan and pazopanib twice a day + oral topotecan. Mice were euthanized when more than 20% body weight loss occurred or when moribund. (A) Mice were imaged every week and tumor growth bioluminescence. i) tumor growth curve,. ii) total body luminescence change over time. (B) Effect on survival. (C) Evaluate the toxicity. i) body weight, ii) WBC count. n=5, bars; +SD

Figure 5. Effect of metronomic oral topotecan alone or in combination with pazopanib

SKOV-3-13 human ovarian cancer cells were injected i.p. into CB-17 SCID mice. Treatments were initiated 14 days after tumor injection. The drug treatments and doses tested were: vehicle control, MTD topotecan (1.5mg/kg i.p. 5 consecutive days every 3 weeks), low-dose metronomic oral topotecan (1mg/kg by gavage, daily), once a day pazopanib (150mg/kg by gavage daily), twice a day pazopanib (25mg/kg by gavage), pazopanib once a day + oral topotecan and pazopanib twice a day + oral topotecan. Mice were euthanized when more than 20% body weight loss occurred or when moribund. (A) Mice were imaged every week and tumor growth bioluminescence. i) tumor growth curve,. ii) total body luminescence change over time. (B) Effect on survival. (C) Evaluate the toxicity. i) body weight, ii) WBC count. n=5, bars; +SD